The invention relates to compositions and methods for the treatment of cancer and other proliferative disorders.
Extracellular signals received at transmembrane receptors are relayed into the cells by the signal transduction pathways (Pelech et al., Science 257:1335 (1992)) which have been implicated in a wide array of physiological processes such as induction of cell proliferation, differentiation or apoptosis (Davis et al., J. Biol. Chem. 268:14553 (1993)). The Mitogen Activated Protein Kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular cues into intracellular responses (Nishida et al., Trends Biochem. Sci. 18:128 (1993); Blumer et al., Trends Biochem. Sci. 19:236 (1994)). Many steps of this cascade are conserved, and homologous for MAP kinases have been discovered in different species.
In mammalian cells, the Extracellular-Signal-Regulated Kinases (ERKs), ERK-1 and ERK-2 are the archetypal and best-studied members of the MAPK family, which all have the unique feature of being activated by phosphorylation on threonine and tyrosine residues by an upstream dual specificity kinase (Posada et al., Science 255:212 (1992); Biggs III et al., Proc. Natl. Acad. Sci. USA 89:6295 (1992); Garner et al., Genes Dev. 6:1280 (1992)).
Recent studies have identified an additional subgroup of MAPKs, known as c-Jun NH2-terminal kinases 1 and 2 (JNK-1 and JNK-2), that have different substrate specificities and are regulated by different stimuli (Hibi et al., Genes Dev. 7:2135 (1993)). JNKs are members of the class of stress-activated protein kinases (SPKs). JNKs have been shown to be activated by treatment of cells with UV radiation, pro-inflammatory cytokines and environmental stress (Derijard et al., Cell 1025 (1994)). The activated JNK binds to the amino terminus of the c-Jun protein and increases the protein""s transcriptional activity by phosphorylating it at ser63 and ser73 (Adler et al., Proc. Natl. Acad. Sci. USA 89:5341 (1992); Kwok et al., Nature 370:223 (1994)).
Analysis of the deduced primary sequence of the JNKs indicates that they are distantly related to ERKs (Davis, Trends Biochem. Sci. 19:470 (1994)). Both ERKs and JNKs are phosphorylated on Tyr and Thr in response to external stimuli resulting in their activation (Davis, Trends Biochem. Sci. 19:470 (1994)). The phosphorylation (Thr and Tyr) sites, which play a critical role in their activation are conserved between ERKs and JNKs (Davis, Trends Biochem. Sci. 19:470 (1994)). However, these sites of phosphorylation are located within distinct dual phosphorylation motifs: Thr-Pro-Tyr (JNK) and Thr-Glu-Tyr (ERK). Phosphorylation of MAPKs and JNKs by an external signal often involves the activation of protein tyrosine kinases (PTKs) (Gille et al., Nature 358:414 (1992)), which constitute a large family of proteins encompassing several growth factor receptors and other signal transducing molecules.
Protein tyrosine kinases are enzymes which catalyze a well defined chemical reaction: the phosphorylation of a tyrosine residue (Hunter et al., Annu Rev Biochem 54:897 (1985)). Receptor tyrosine kinases in particular are attractive targets for drug design since blockers for the substrate domain of these kinases is likely to yield an effective and selective antiproliferative agent. The potential use of protein tyrosine kinase blockers as antiproliferative agents was recognized as early as 1981, when quercetin was suggested as a PTK blocker (Graziani et al., Eur. J. Biochem. 135:583-589 (1983)).
The best understood MAPK pathway involves extracellular signal-regulated kinases which constitute the Ras/Raf/MEK/ERK kinase cascade (Boudewijn et al., Trends Biochem. Sci. 20, 18 (1995)). Once this pathway is activated by different stimuli, MAPK phosphorylates a variety of proteins including several transcription factors which translocate into the nucleus and activate gene transcription. Negative regulation of this pathway could arrest the cascade of these events.
What are needed are new anticancer chemotherapeutic agents which target receptor tyrosine kinases and which arrest the Ras/Raf/MEK/ERK kinase cascade. Oncoproteins in general, and signal transducing proteins in particular, are likely to be more selective targets for chemotherapy because they represent a subclass of proteins whose activities are essential for cell proliferation, and because their activities are greatly amplified in proliferative diseases.
What is also needed are new cell antiproliferative agents, and anticancer therapeutics in particular, which are highly selective in the killing of proliferating cells such as tumor cells, but not normal cells.
According to one embodiment of the invention, novel compounds are provided according to formula I: 
wherein
R1 and R2 are independently selected from the group consisting of chlorine, fluorine and bromine; and
R3 is selected from the group consisting of hydrogen and fluorine;
R1 and R2 may not both be chlorine when R3 is hydrogen; and
R1 may not be chlorine when R2 is fluorine and R3 is hydrogen in the same compound.
According to another embodiment of the invention, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a compound of formula II 
wherein
n is zero or one;
R1 is selected from the group consisting of hydrogen, chlorine, fluorine and bromine;
R2 is selected from the group consisting of hydrogen, chlorine, fluorine, bromine, methyl and methoxy; and
R3 is selected from the group consisting of hydrogen, chlorine and fluorine; provided,
R2 may not be methyl or methoxy when R1 and R3 are both hydrogen and n is zero or one; and
R1, R2 and R3 may not all be hydrogen when n is one.
According to a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a compound of the formula II, wherein R3 is hydrogen, and R1 and R2 are independently selected from the group consisting of chlorine, fluorine and bromine.
According to another embodiment of the invention, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a compound of the formula III 
wherein R1 is selected from the group consisting of hydrogen, chlorine, fluorine and bromine.
The invention also relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the formula IV wherein R1 is selected from the group consisting of fluorine and bromine, and 
R2 is selected from the group consisting of 2-chlorophenyl, 4-chlorophenyl, 4-fluorophenyl and 2-nitrophenyl.
According to another embodiment of the invention, a method of treating an individual for a proliferative disorder, particularly cancer, is provided comprising administering to said individual an effective amount of a compound according to any of formulae II, III or IV, alone or in combination with a pharmaceutically acceptable carrier. In another embodiment, a method of inhibiting growth of tumor cells in an individual afflicted with cancer is provided, comprising administering to said individual an effective amount of a compound according to formula II, III or IV, alone or in combination with a pharmaceutically acceptable carrier. Furthermore, a method of inducing apoptosis of tumor cells in an individual afflicted with cancer is provided comprising administering to said individual an effective amount of a compound according to formula II, III or IV, alone or in combination with a pharmaceutically acceptable carrier.
According to certain embodiments of the invention, the treatment is for breast or prostate cancer, and the cells whose growth is inhibited are breast or prostate tumor cells. According to another embodiment of the invention, the treatment is for a proliferative disorder, other than breast and prostate cancer, and the cells whose growth is inhibited are other than breast or prostate tumor cells. According to certain embodiments of the invention, the cancers treated are selected from the group consisting of cancers of the ovaries, lung, colon, rectum, brain, kidney, pancreas and skin; leukemias; lymphomas; sarcomas; squamous cell carcinomas and papillomas; basal cell carcinomas and papillomas; and epidermoid cancers. The growth of cancer cells, particularly tumor cells associated with these cancers, is inhibited. The cells are induced to undergo apoptosis.